The germinal center of mammals comprises a highly specialized microenvironment required for the final process of maturation towards antigen specific memory cells and long-lived plasma cells (Embo J., 16:2996-3006, 199; Semin. Immunol., 4:11-17, 1992). In this microenvironment, two fundamental editings of the immunoglobulin genes take place (J. Exp. Med., 173:1165-1175, 1991; Embo. J., 12:4955-4967, 1993; Adv. Exp. Med. Biol., 186:145-151, 1985; Nature, 342:929-931, 1989; Cell, 67:1121-1129).
The first fundamental editing is somatic hypermutation (Curr. Opin. Immunol., 7:248-254, 1995; Annu. Rev. Immunol., 14:441-457, 1996; Science, 244:1152-1157, 1989), a phenomenon in which extensive point mutation in the exons of genes encoding variable regions of immunoglobulins occurs. Accumulation of point mutations leads to selection of B cells expressing high affinity immunoglobulins on their cell surface, accompanied by the affinity maturation of antibodies (Embo. J., 4:345-350, 1985; Proc. Natl. Acad. Sci. USA, 85:8206-8210, 1988). As a result, immunoglobulin genes are edited as new functional genes.
Another fundamental editing process is the class switch recombination (CSR). In CSR, effector functions of antibodies, such as complement fixation, are selected by exchanging exons encoding constant regions of immunoglobulin heavy chains (Curr. Top. Microbiol. Immunol., 217:151-169, 1996; Annu. Rev. Immunol., 8:717-735, 1990).
These two types of genetic editing are very important for effective humoral immunoreaction to eliminate harmful microbes. The molecular mechanisms of the genetic phenomena have not yet been elucidated despite extensive study for several decades.
The present inventors isolated a mouse B cell clone, CH12F3-2, as a research tool to elucidate the molecular mechanism of class switch recombination of immunoglobulin. In this B cell line, class switch recombination (CSR) from IgM to IgA begins several hours after stimulation with IL-4, TGF-β, and CD40L; ultimately, over 80% of the cells become IgA positive (Immunity, 9:1-10, 1998; Curr. Biol., 8:227-230, 1998; Int. Immunol., 8:193-201, 1996).
Using the mouse B cell clone CH12F3-2, the present inventors previously reported that the breakpoints of CSR distribute not only in the switch region (or “S region”), characterized by repeated sequences, but also in neighboring sequences (Curr. Biol., 8:227-230, 1998). However, the breakpoints were rarely seen in I exon and C exon, which are located upstream and downstream of the S region, respectively. Also, according to accumulated scientific evidence, it has been shown that transcription of I exon and C exon and splicing of the transcripts are essential for CSR (Cell, 73:1155-1164, 1993; Science, 259:984-987, 1993; Proc. Natl. Acad. Sci, USA, 90:3705-3709, 1993; Cell, 81:833-836, 1995).
This suggests that the transcripts are involved in CSR either directly or indirectly. Accordingly, the present inventors propose a theory that class switch is initiated by the recognition of DNA-RNA complex structure and not by the recognition of nucleotide sequences of the switch region. This idea is further fortified by the fact that even when the Sa region is substituted with an Sα region or an Sγ region by introducing a mini-chromosome into the above-mentioned mouse B cell clone CH12F3-2, CSR in the mini-chromosome efficiently occurs after stimulation with cytokines (Immunity, 9:1-10, 1998).
In plants and protozoa, RNA editing, another type of genetic editing, is widely used as a mean for producing functional genes from a limited genome (Cell, 81:833-836, 1995; Cell, 81:837-840, 1995). mRNA editing of many molecules such as the mRNA for apolipoprotein B (apoB), AMPA receptors, Wilmstumor-1, α-galactosidase and neurofibromatosis type-1, and tRNA-Asp, have been reported (Trends Genet., 12:418-424, 1996; Curr. Opin. Genet. Dev., 6:221-231, 1996). Although the molecular mechanism of mammalian RNA editing has not yet been elucidated, one performed by APOBEC- 1 (apolipoprotein B mRNA editing enzyme, catalytic polypeptide-1) is becoming understood by degrees (Science, 260:1816-1819, 1993; J. Biol. Chem., 268:20709-20712, 1993).
In apoB RNA editing, the first base C (cytosine) of codon CAA, which encodes glutamine, is converted to U (uridine), which alters the codon to UAA. As a result, an in-frame stop codon is made in the apoB mRNA (J. Cell., 81:187-195, 1995; J. Cell., 50:831-840, 1987; Science, 238:363-266, 1987). apoB-48 and apoB-100 are transcripts of edited mRNA and unedited mRNA of apoB, respectively, and these proteins possess totally different physiological functions from each other (J. Biol. Chem., 271:2353-2356, 1996).
In site-specific RNA-editing, auxiliary factors are required (Science, 260:1816-1819, 1993; J. Biol. Chem., 268:20709-20712, 1993). In the absence of auxiliary factors, APOBEC-1 shows only a cytidine deaminase activity, possessing non-specific low affinity to RNA(J. Biol. Chem., 268:20709-20712, 1993; J. Cell., 81:187-195, 1995; J. Biol. Chem., 270:14768-14775, 1995; J. Biol. Chem., 270:14762-14767, 1995). The expression and activity of the auxiliary factors are found not only in organs with apoB mRNA editing, but also in organs with undetectable levels of APOBEC-1 expression, or organs without apoB mRNA editing (Science, 260:1816-1819, 1993; J. Biol. Chem., 268:20709-20712, 1993; Nucleic Acids Res., 22:1874-1879, 1994; Proc Natl. Acad. Sci, USA, 91:8522-8526, 1994; J. Biol. Chem., 269:21725-21734, 1994).
The unexpected expression of the auxiliary factors involved in apoB mRNA editing suggests that the auxiliary factors may be involved in more general cellular functions or other yet unknown RNA editing. Since the possibility exists that CSR and hypermutation, which are involved in genetic editing of immunoglobulin genes, may be accomplished by RNA editing, it would be very interesting to elucidate whether RNA editing takes place or not in the genetic editing of immunoglobulin genes as mentioned above.